Sulphasalazine inhibits macrophage activation: inhibitory effects on inducible nitric oxide synthase expression, interleukin-12 production and major histocompatibility complex II expression

Abstract

The anti-inflammatory agent sulphasalazine is an important component of several treatment regimens in the therapy of ulcerative colitis, Crohn's disease and rheumatoid arthritis. Sulphasalazine has many immunomodulatory actions, including modulation of the function of a variety of cell types, such as lymphocytes, natural killer cells, epithelial cells and mast cells. However, the effect of this agent on macrophage (Mφ) function has not been characterized in detail. In the present study, we investigated the effect of sulphasalazine and two related compounds – sulphapyridine and 5-aminosalicylic acid – on Mφ activation induced by bacterial lipopolysaccharide (LPS) and interferon-γ (IFN-γ). In J774 Mφ stimulated with LPS (10 µg/ml) and IFN-γ (100 U/ml), sulphasalazine (50–500 µm) suppressed nitric oxide (NO) production in a concentration-dependent manner. The expression of the inducible NO synthase (iNOS) was suppressed by sulphasalazine at 500 µm. Sulphasalazine inhibited the LPS/IFN-γ-induced production of both interleukin-12 (IL-12) p40 and p70. The suppression of both NO and IL-12 production by sulphasalazine was superior to that by either sulphapyridine or 5-aminosalicylic acid. Although the combination of LPS and IFN-γ induced a rapid expression of the active forms of p38 and p42/44 mitogen-activated protein kinases and c-Jun terminal kinase, sulphasalazine failed to interfere with the activation of any of these kinases. Finally, sulphasalazine suppressed the IFN-γ-induced expression of major histocompatibility complex class II. These results demonstrate that the Mφ is an important target of the immunosuppressive effect of sulphasalazine.

Introduction

Sulphasalazine is a complex anti-inflammatory agent synthesized by linking the sulphonamide antibiotic sulphapyridine to 5-aminosalicylic acid.¹ Sulphasalazine is the mainstay of therapy in the treatment of inflammatory bowel disease, which manifests either as ulcerative colitis or Crohn's disease.¹ Sulphasalazine is also used as a second-line agent in the treatment of rheumatoid arthritis, where it is often combined with methotrexate, which is the most commonly prescribed disease-modifying antirheumatic drug.² After oral administration, 10–20% of the sulphasalazine is absorbed from the small intestine. The remaining 80–90% reaches the colon, where it is cleaved by colonic bacteria into its two moieties: sulphapyridine and 5-aminosalicylic acid. While the & non-absorbable 5-aminosalicylic acid appears to be the active moiety in inflammatory bowel disease, the sulphapyridine part is thought to be the active metabolite in rheumatoid arthritis.

Sulphasalazine exerts a wide variety of immunosuppressive effects targeting distinct cell populations, such as epithelial cells,³ neutrophils,⁴ T and B lymphocytes,^5,6 mast cells,⁷ natural killer (NK) cells⁸ and endothelial cells.⁹ The immunosuppressive effects of sulphasalazine include suppression of cytokine production,¹⁰ chemotaxis,¹¹ adhesion molecule expression,¹² antibody production⁵ and free radical scavenging.¹ Although there is some evidence demonstrating that sulphasalazine inhibits the production of tumour necrosis factor-α (TNF-α), interleukin (IL)-1 and IL-6 in mononuclear cells,¹⁰ its effect on macrophages (Mφ) is less well understood. Stimulation of Mφ induces the expression of a large number of genes, which results in the synthesis of enzymes able to produce soluble extracellular mediators such as nitric oxide (NO) or arachidonate metabolites, and the production of proteins, which are directly involved in intercellular communication. These latter include cytokines, adhesion molecules and histocompatibility antigens. NO produced by the inducible NO synthase (iNOS) in immunostimulated Mφs, is one of the major players in causing tissue injury in both inflammatory bowel disease^13,14 and rheumatoid arthritis.¹⁵ While NO, TNF-α, IL-1 and IL-6 are directly involved in the development of tissue injury, the early production of IL-12 is the driving force of the immune/inflammatory process. IL-12 stimulates T cells and NK cells to proliferate and produce interferon-γ (IFN-γ), which in turn activates effector cells to produce cytotoxic products, such as cytokines and NO. In the current study, we investigated whether and how sulphasalazine and its breakdown products affect NO and IL-12 production in Mφs. Furthermore, as major histocompatibility complex (MHC) class II up-regulation is an important measure of Mφ activation, we examined the effect of sulphasalazine on IFN-γ-induced up-regulation of MHC class II molecules.

Materials and Methods

Drugs and reagents

Lipopolysaccharide (LPS) (from Escherichia coli 055 : B5), sulphanilamide, naphthylethylenediamide, thioglycollate medium, dimethylsulphoxide, sulphasalazine, sulphapyridine and 5-aminosalicylic acid were purchased from Sigma (St. Louis, MO). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Fisher Scientific (Pittsburgh, PA). IFN-γ was obtained from R & D Systems (Minneapolis, MN). RPMI-1640 was purchased from Life Technologies (Grand Island, NY).

Culture of J774.1 Mφs

The mouse Mφ cell line J774.1 was grown in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin and 100 µg/ml of streptomycin in a humidified atmosphere of 95% air and 5% CO₂.

Treatment of J774 Mφs for NO and IL-12 measurement

Cells were cultured in 96-well plates (200 µl of medium per well) until reaching 80% confluence. The cells were treated with sulphasalazine, sulphapyridine, or 5-aminosalicylic acid 30 min before induction with a combination of LPS (10 µg/ml) and IFN-γ (0·5 µg/ml). Supernatants were harvested 24 hr after stimulation with LPS/IFN-γ.

Measurement of nitrite concentration

Nitrite production, an indicator of NO synthesis, was measured, as previously described,¹⁶ by adding 100 µl of Griess reagent (1% sulphanilamide and 0·1% naphthylethylenediamide in 5% phosphoric acid) to 100-µl samples of medium. The absorbance at 550 nm (A₅₅₀) was measured using a Spectramax 250 microplate reader (Molecular Devices, Sunnyvale, CA). The detection limit of the assay is 1 µm. The measurements of nitrite/nitrate were performed using reagents free of nitrite and nitrate: no basal or background nitrite or nitrate levels were detected. Using a standard curve for nitrite (10–200 µm), we found that sulphasalazine (from 10 to 500 µm) caused a reduction of ≈ 15% in the A₅₅₀, indicating that sulphazalazine interferes with the Griess reaction. When calculating the effect of sulphasalazine on NO production, we corrected the Griess reaction for this interference. We achieved this by multiplying the percentage inhibition caused by sulphasalazine by 0·85.

Cytokine assays

Cytokine concentrations in the supernatants were determined by enzyme-linked immunosorbent assay (ELISA) kits that are specific against murine cytokines. Levels of IL-12 p40 and IL-12 p70 were measured using ELISA kits purchased from Genzyme Co. (Boston, MA). Plates were read at 450 nm by using a Spectramax 250 microplate reader from Molecular Devices. Detection limits were 10 pg/ml for IL-12 p40 and 5 pg/ml for IL-12 p70. Assays were performed as described previously¹⁷ and according to the manufacturer's instructions.

Measurement of mitochondrial respiration

Mitochondrial respiration, an indicator of cell viability, was assessed by the mitochondrial-dependent reduction of MTT to formazan.¹⁶ Cells in 96-well plates were incubated with MTT (0·2 mg/ml) for 60 min at 37°. Culture medium was removed by aspiration and the cells were solubilized in dimethylsulphoxide (100 µl). The extent of reduction of MTT to formazan within the cells was quantified by measurement of the A₅₅₀ using a Spectramax microplate reader (Molecular Devices).

Western blot analysis

Cells in six-well plates were pretreated with sulphasalazine or vehicle, and 30 min later the cells were stimulated with LPS (10 µg/ml) and IFN-γ (0·5 µg/ml) for 15 min for determination of activation of mitogen-activated protein kinase (MAPK, ERK 1/2, p42/44), c-Jun N-terminal protein kinase (JNK) and p38. For the measurement of iNOS induction, the cells were treated and stimulated in a similar manner as for MAPK measurements, and were harvested 6 hr after stimulation. After washing with phosphate-buffered saline (PBS), the cells were lysed by the addition of modified radioimmunoprecipitation buffer (50 mm Tris-HCl, 150 mm NaCl, 1 mm EDTA, 0·25% Na-deoxycholate, 1% Nonidet P-40 [NP-40], 1 µg/ml of pepstatin, 1 µg/ml of leupeptin, 1 mm phenylmethylsulphonyl fluoride [PMSF], 1 mm Na₃VO₄). The lysates were transferred to Eppendorf tubes, centrifuged at 15 000 g and the supernatant was recovered. Protein concentrations were determined using a Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Ten micrograms of sample was separated on a 8–16% Tris-Glycine gel (Novex, San Diego, CA) and transferred to a nitrocellulose membrane. The membranes were probed with anti-phospho-MAPK (p42/p44), anti-phospho-JNK (Promega, Madison, WI), anti-phospho-p38 (New England Biolabs, Beverly, MA), or anti-iNOS (Calbiochem, San Diego, CA), and subsequently incubated with a secondary horseradish peroxidase (HRP)-conjugated donkey anti-rabbit antibody (Boehringer, Indianapolis, IN). After performing the iNOS Western blotting, the membrane was stripped and reprobed with an antibody against actin (Santa Cruz Biotechnology, Santa Cruz, CA) Bands were detected using the ECL Western Blotting Detection Reagent (Amersham Life Science, Arlington Heights, IL).

Preparation and treatment of peritoneal Mφs

Male BALB/c mice were injected intraperitoneally with 2 ml of 2% thioglycollate, and peritoneal cells were harvested 3–4 days later. The cells were plated onto 12-well plastic plates at 1 × 10⁶ cells/ml and incubated in RPMI-1640 (Life Technologies) for 2 hr at 37° in a humidified 5% CO₂ incubator. Non-adherent cells were removed by rinsing the plates three times with 5% dextrose in PBS.

Detection of surface I-A^d by flow cytometry

Peritoneal Mφs were treated with increasing concentrations of sulphasalazine, in the presence or absence of IFN-γ (0·5 µg/ml), for 48 hr. Cells were removed by scraping into 0·5 ml of Versene (Life Technologies) and washed in PBS. After washing, the cells were resuspended in PBS containing 10% mouse serum and Fc Block (rat anti-mouse CD16/CD32; Pharmingen, San Diego, CA) and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-I-A^d (Pharmingen). The cells were analysed by using a fluorescence-activated cell sorter (FACSCalibur; Becton-Dickinson Immunocytometry Systems, San Jose, CA).

Results

Effect of sulphasalazine, sulphapyridine and 5-aminosalicylic acid on NO production in J774 Mφs

The combination of LPS (10 µg/ml) and IFN-γ (0·5 µg/ml), but neither agent alone, induced the production of NO (the nitrite concentration was 137 ± 3·5 µm, n = 6). Pretreatment of cells with sulphasalazine 30 min before stimulation with LPS/IFN-γ caused a concentration-dependent reduction in NO production, which reached statistical significance at 100 µm (Fig. 1a). The suppression of NO production reached 42% at 500 µm sulphasalazine. Sulphasalazine was not toxic to the cells at concentrations up to 500 µm (Fig. 1b). Higher doses of sulphasalazine, however, were toxic, and therefore their effect on NO production was not determined. Figure 1(c) shows that 5-aminosalycilic acid caused a slight, but significant, suppression of NO production at 5000 µm. However, sulphapyridine was without effect (Fig. 1d). Neither 5-aminosalycilic acid nor sulphapyridine were toxic to the cells at the concentrations tested (data not shown).

Figure 1.

Effect of sulphasalazine (a), 5-aminosalicylic acid (c) and sulphapyridine (d) on nitric oxide (NO) production in J774 cells induced by lipopolysaccharide (LPS) (10 µg/ml) and interferon-γ (IFN-γ) (0·5 µg/ml). Figure 1(b) shows that sulphasalazine is not toxic to the cells up to a concentration of 500 µm, as determined with the (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were pretreated with sulphasalazine 30 min before stimulation with LPS/IFN-γ, and NO concentrations were determined from the supernatants 24 hr after stimulation with LPS/IFN-γ. Data are expressed as the mean ±SEM of six wells. *P < 0·05; **P < 0·01.

Effect of sulphasalazine on iNOS expression in J774 Mφs

To investigate whether the decrease in NO production by sulphasalazine was the result of a decrease in iNOS formation, we determined the effect of sulphasalazine on iNOS protein expression. The combination of LPS (10 µg/ml) and IFN-γ (0·5 µg/ml) induced the appearance of iNOS protein, as determined by Western blotting 6 hr after stimulation. Pretreatment of the cells with 500 but not 100 µm sulphasalazine 30 min before stimulation suppressed iNOS expression (Fig. 2). The inhibitory effect of 500 µm sulphasalazine on iNOS expression was selective, because 500 µm sulphasalazine did not decrease the expression of actin (Fig. 2).

Figure 2.

Sulphasalazine suppresses the expression of inducible nitric oxide synthase (iNOS), but not actin, in J774 cells induced by lipopolysaccharide (LPS) (10 µg/ml) and interferon-γ (IFN-γ) (0·5 µg/ml). The cells were pretreated with sulphasalazine 30 min before stimulation with LPS/IFN-γ. Cell extracts were prepared 12 hr after stimulation and iNOS or actin expression was determined by Western blotting. Lanes 1 and 2, no stimulation; lanes 3 and 4, stimulation with 10 µg/ml of LPS and 0·5 µg/ml of IFN-γ; lane 5, 100 µm sulphasalazine pretreatment 30 min before stimulation with LPS/IFN-γ; lane 6, 500 µm sulphasalazine pretreatment 30 min before stimulation with LPS/IFN-γ. This blot is representative of four separate experiments.

Effect of sulphasalazine, sulphapyridine and 5-aminosalicylic acid on IL-12 production in J774 Mφs

The combined administration of LPS (10 µg/ml) and IFN-γ (0·5 µg/ml) to J774 cells for 24 hr caused the appearance of IL-12 p40 in the culture supernatants. Pretreatment of the cells with sulphasalazine 30 min before stimulation suppressed IL-12 p40 production, in a dose-dependent manner (Fig. 3; 50% effective concentration [EC₅₀] ≈ 150 µm). However, both sulphapyridine and 5-aminosalicylic acid were much less potent, as 5000 µm 5-aminosalicylic acid and 3000 µm sulphapyridine were needed for a significant suppression of p40 (Table 1). Incubation of J774 cells with LPS (10 µg/ml) and IFN-γ (0·5 µg/ml) for 24 hr also induced the production of IL-12 p70, which was suppressed by pretreatment of the cells with sulphasalazine. The IL-12 p70 level in LPS/IFN-γ-treated cells was 32·18 ± 1·27 pg/ml (n = 4) compared to 21·93 ± 2·76 pg/ml (n = 4) in the sulphasalazine-pretreated cells (P < 0·05).

Figure 3.

Sulphasalazine suppresses interleukin-12 (IL-12) p40 production by J774 cells stimulated with lipopolysaccharide (LPS) (10 µg/ml) and interferon-γ (IFN-γ) (0·5 µg/ml). Sulphasalazine was administered 30 min before stimulation with LPS/IFN-γ. The level of IL-12 p40 was determined from the supernatants 24 hr after stimulation. Data are expressed as the mean ±SEM of six wells. *P < 0·05; **P < 0·01.

Table 1.

Effect of different concentrations of 5-aminosalicylic acid (5-ASA) and sulphapyridine on lipopolysaccharide/interferon-γ (LPS/IFN-γ)-stimulated interleukin-12 (IL-12) production by J774 macrophages (Mφs)

Drug	IL-12 (% of control)
Control	100·0 ± 5·7
100 µm 5-ASA	90·0 ± 8·1
500 µm 5-ASA	97·0 ± 13
1000 µm 5-ASA	88·0 ± 10
5000 µm 5-ASA	54·0 ± 3·2**
100 µm sulphapyridine	97·0 ± 4·9
300 µm sulphapyridine	99·0 ± 9·8
100 µm sulphapyridine	102·0 ± 7·1
3000 µm sulphapyridine	66·6 ± 3·8**

J774 macrophages (Mφs) were pretreated with 5-ASA or sulphapyridine 30 min before stimulation with LPS (10 µg/ml) and IFN-γ (0·5 µg/ml), and IL-12 p40 concentrations were determined from the supernatants 24 hr after stimulation with LPS/IFN-γ. Data are expressed as the mean ±SEM of six wells.

^**P < 0·01.

Sulphasalazine fails to influence LPS-induced activation of the p38 and p42/44 MAPKs, or the phosphorylation of JNK

Because activation of the MAPKs p38 and p42/44 and the phosphorylation of JNK are important pathways during Mφ activation,^18–20 we tested the possibility that sulphasalazine exerts its effects on NO and IL-12 production via interfering with the activation of these enzymes. Although LPS/IFN-γ treatment of peritoneal Mφs caused the activation of both p38 and p42/44 as well as JNK (determined 15 min after LPS/IFN-γ treatment) sulphasalazine administered 30 min before the LPS/IFN-γ challenge failed to alter the activation of these intracellular pathways (Fig. 4).

Figure 4.

Sulphasalazine fails to affect p42/44 mitogen-activated protein kinase (MAPK) (ERK1/2) and c-Jun N-terminal protein kinase (JNK) activation or the phosphorylation of p38 in J774 cells. Lanes 1 and 2, no stimulation; lanes 3 and 4, 10 µg/ml of lipopolysaccharide (LPS) and 0·5 µg/ml of interferon-γ (IFN-γ); lanes 5 and 6, sulphasalazine pretreatment 30 min before stimulation with LPS/IFN-γ. The activation of p42/44, JNK and p38 was determined from cellular extracts obtained 15 min after stimulation with LPS/IFN-γ. The results are representative of two different experiments.

Sulphasalazine suppresses IFN-γ-induced up-regulation of surface I-A^d molecules in peritoneal Mφs

To further examine the effect of sulphasalazine on Mφ activation, we measured surface expression of MHC class II molecules in response to IFN-γ. Peritoneal Mφs were used, because IFN-γ failed to induce up-regulation of MHC class II in J774 cells (data not shown). I-A^d expression (MHC class II in BALB/c mice) was decreased, in a concentration-dependent manner, by treatment with sulphasalazine (Fig. 5).

Figure 5.

Sulphasalazine suppresses the expression of major histocompatibility complex (MHC) class II antigens on peritoneal cells stimulated with interferon-γ (IFN-γ) for 48 hr, as determined by flow cytometry. (a) Scattergram of peritoneal macrophages (Mφs). (b) Mean fluorescence of non-stimulated cells. (c) Mean fluorescence of cells exposed to interferon-γ (IFN-γ) (0·5 µg/ml) for 48 hr. (d) Mean fluorescence of cells pretreated with 100 µm sulphasalazine 30 min before stimulation with IFN-γ. (e) Mean fluorescence of cells pretreated with 500 µm sulphasalazine 30 min before stimulation with IFN-γ. This figure is representative of two different experiments.

Discussion

Activated Mφs play an important role in the induction of tissue injury in autoimmune/inflammatory diseases, such as inflammatory bowel disease and rheumatoid arthritis. The role of Mφs in autoimmune disease is twofold. First, Mφs are central to the initiation of disease. They function by shaping the immune response both as antigen-presenting cells, where their function is crucially dependent on IL-12 production,^21,22 as well as by affecting the expression of cell-surface molecules, such as MHC class II²³ and the B7 costimulatory molecules.²⁴ Second, Mφs are also the terminal effector cells of inflammation as they produce both cytokines and free radicals capable of causing direct injury to the intestinal mucosa²⁵ or synovial tissue.²⁶ Our findings suggest that sulphasalazine acts to suppress Mφ functions associated with both the initiation and maintenance of an immune/inflammatory response. The fact that sulphasalazine suppresses IL-12 production (this work and ref. 27) and MHC class II expression underlines the importance of introducing novel therapeutic regimens aiming at an early administration of sulphasalazine in rheumatoid arthritis.² It is also important to note that sulphasalazine was more potent than either 5-aminosalicylic acid or sulphapyridine in suppressing IL-12 production. These differences in potency may explain why sulphasalazine is superior to either of its moieties in the treatment of autoimmune diseases.

Another important finding of the current study is that sulphasalazine suppresses NO production. The pathophysiological role of NO in rheumatoid arthritis has been amply documented.^15,28,29 The issue of such a role for iNOS in inflammatory bowel disease is controversial. That is, although it was shown that genetic³⁰ inhibition of iNOS is detrimental, the majority of the pharmacological studies support the notion that iNOS inhibition is beneficial in animal models of inflammatory colitis.^13,14 Our demonstration that sulphasalazine suppresses NO production by Mφs suggests that such an effect may contribute to the therapeutic effect of sulphasalazine in inflammatory bowel disease and rheumatoid arthritis. Interestingly, sulphasalazine failed to suppress increased plasma NO levels in spontaneous colitis observed in HLA-B27 transgenic rats.³¹ However, plasma NO levels may not reflect the local levels of NO in the inflamed tissue. Furthermore, the effect of sulphasalazine on NO may be cell specific, as sulphasalazine failed to inhibit NO production by chondrocytes.³² The discrepancy observed in our study between the lower potency of sulphasalazine in suppressing iNOS expression (500 µm), as compared to NO production (100 µm), may be the result of an effect on the activity of iNOS. This notion is supported by the findings of Reynolds et al.³³ who showed that sulphasalazine suppresses NO production in a cell-free system. As sulphasalazine concentrations can reach as high as 5 mm in the gut,¹ our findings have clinical relevance.

Based on our findings that sulphasalazine suppressed the expression of IL-12, iNOS and MHC class II, which are all dependent on the transcription factor nuclear factor-κB (NF-κB), plus earlier data from another group demonstrating the ability of sulphasalazine to inhibit activation of this transcription factor,³ it can be proposed that the mechanisms by which sulphasalazine suppresses Mφ activation involve an effect on NF-κB. While NF-κB is a possible target of the action of sulphasalazine, activation of the MAPKs p38 and p42/44 and the phosphorylation of JNK does not appear to be affected by sulphasalazine. Further studies will be needed to determine the mode of action of sulphasalazine at the cellular level.

In conclusion, our findings demonstrate that sulphasalazine suppresses Mφ activation, which may explain some of the anti-inflammatory effects of this agent.